Transmission of extracellular information into the cell in most cases requires mediation by membrane proteins that have transmembrane domains. G protein-coupled receptors (GPCRs) are signal-transmitting membrane proteins that have seven transmembrane domains and make up a receptor family that can bind various physiological peptide ligands, including biological amines such as dopamine and serotonin, lipid derivatives such as prostaglandin, nucleic acids such as adenosine, amino acids such as GABA, angiotensin II, bradykinin, and cholecystokinin. Serving also as receptors for extracellular transmitters responsible for the senses of vision, taste and smell, GPCRs are important membrane proteins that play a key role in signal transduction.
The recent progress in completing the human genome sequence is expected to lead to discovery of many orphan receptors that are suspected of being GPCR. If successfully identified, the ligands for these GPCRs will allow for more effective development of pharmaceutical products. Thus, devising a structural model for G protein-coupled receptor/ligand complexes and devising a three-dimensional structural model for G protein-coupled receptors in the structural model of the complexes will provide an important approach to the future development of pharmaceutical products, as will the identification, screening, searching, evaluation, and designing methods of ligands that take advantage of these structural models.
In fact, a number of patent applications entitled “novel G protein-coupled receptor protein and its DNA” have recently been filed, including Japanese Laid-Open Patent Publications No. 2001-29083, No. 2001-29084, No. 2001-54388, No. 2001-54389, No. 2000-23676, No. 2000-23677, No. 2000-50875, No. 2000-152792, No. 2000-166576, No. 2000-175690, No. 2000-175691, and No. 2000-295995, to name a few. Some applications, such as Japanese Patent Laid-Open Publication No. 2000-354500, disclose methods for screening for ligands that bind to G protein-coupled receptors while other applications concern methods for cloning expression of G protein-coupled receptors.
Ligands that bind to a particular GPCR are generally classified into agonists and antagonists. According to the latest pharmacological classification standards, the former is further divided into full agonists and partial agonists and the latter into inverse agonists and antagonists.
These ligands are classified not by their affinity for the receptor, but by the degree to which the ligand activates the receptor. For example, assuming the activity elicited by binding of a full agonist to be 100%, a partial agonist elicits a 50 to 70% activity.
In comparison, binding of an antagonist suppresses the activity to 5 to 10% of what is elicited by the binding of a full agonist, and binding of an inverse agonist completely eliminates the activity (0% activity).
Even when unbound to ligands, many GPCRs exhibit 5 to 10% of the activity expected by the binding of a full agonist. Thus, it is believed that antagonists bind to physiologically inactive receptor conformations. This suggests that binding of other types of ligands brings about conformational change of GPCR. Thus, the binding of ligands and subsequent conformational change of receptors are believed to play an important role in information transmission mediated by GPCR.
G protein-coupled receptors (GPCR), which share seven transmembrane domains, are classified into different families based on the homology of their amino acid sequences. In one such GPCR family, each member has high homology to rhodopsin, a photoreceptor membrane protein. The GPCRs of this family share highly conserved amino acid residues in their transmembrane domains. These amino acid residues are believed to play an important role in the functioning of GPCRs.
Structural and functional studies of GPCR have been conducted by analyzing three-dimensional structure of rhodopsin through two-dimensional cryoelectron diffraction crystallography and X-ray crystallography (Palczewski, K. et al., Science 289, 739-745. (2000)). Also, structures of the receptor proteins and the chromophores to serve as ligands, as well as the receptors' conformational changes, have been studied using FT-IR and Raman spectroscopy (Sakmar, T. P., Prog. Nucleic Acid Res. 59, 1-34 (1998)).
Based on the results of two-dimensional, low-resolution, cryoelectron diffraction crystallography, a three-dimensional structural model of rhodopsin was first constructed. More recently, more detailed three-dimensional structure of rhodopsin was revealed by X-ray crystallography. This structure was consistent with the structural characteristics previously expected from the results of FT-IR and Raman spectroscopy and made it possible to formulate assumptions about the roles of some parts of the highly conserved amino acid residues of GPCRs.
For example, of the highly conserved amino acid residues of rhodopsin, the Glu134-Arg135-Tyr136 triplet (ERY triplet, which corresponds to Asp-Arg-Tyr, or DRY triplet, in other GPCRs) of the third transmembrane helix (TM3) (hereinafter, each of the seven transmembrane helices may be denoted by abbreviation followed by respective consecutive numbers: n th helix is denoted as TMn (e.g., TM3)) located on the inside of the cell plays a significant role in the activation of G protein. It has been shown that the protonation of ionized Glu134 in metarhodopsin II (described later), an activated conformation of rhodopsin, triggers activation of G-protein (Arnis, S. & Hofmann, K. P., Proc. Natl. Acad. Sci. USA, 90, 7849-7853, 1993). Also, a significant involvement of Glu and Arg in the activation of GPCRs is suggested.
On the other hand, it is suggested that a highly conserved Pro residue found in TM6 and TM7 (Pro 267 in TM6) is responsible for the kink structure characteristic of these two helices. However, the role of the kink in the functioning of GPCRs still remains unclear.
Hydrophilic amino acid residues Asn55, Asp83, Asn302 found in TM1, TM2, and TM7, respectively, are linked to one another via hydrogen bonds. Also, Tyr306 residue conserved among TM7s is linked, through hydrophobic interaction, to a residue of C-terminal helix located on the inside of the cell. These interactions are believed to contribute to stabilizing the structure.
Rhodopsin is also one of the GPCRs closely studied for its conformational change and functions. Rhodopsin consists of 11-cis-retinal, a chromophore, and opsin, a protein component with the seven transmembrane domains. 11-cis-retinal is covalently bonded to Lys296 to form a Schiff base. This Schiff base is protonated and is thus responsible for the shift of the maximum UV absorbance (λmax) of the chromophore to a long-wavelength range of 498 nm.
When illuminated, rhodopsin is converted to highly unstable bathorhodopsin (which may be referred to simply as ‘Batho,’ hereinafter), which has the UV absorbance shifted to an even longer wavelength range. Upon this, 11-cis-retinal is converted to 11-trans-retinal, an all-trans chromophore. The unstable, high-energy Batho is then sequentially converted to different intermediates in the order of lumirhodopsin (‘Lumi,’ hereinafter), metarhodopsin I (‘Meta I,’ hereinafter), metarhodopsin Ib (‘Meta Ib,’ hereinafter), and metarhodopsin II (‘Meta II,’ hereinafter) as the chromophore and opsin thermally undergo conformational changes (Tachibanaki, S. et al., Biochemistry 36, 14173-14180 (1997)) (the photoreaction process is shown in FIG. 1).
Under physiological conditions, Lumi is converted to Meta II via an intermediate known as metarhodopsin I380 (‘Meta I380,’ hereinafter) (Thorgeirsson, T. E. et al., Biochemistry 32, 13861-13872 (1993)) (FIG. 1).
Because the activation of G protein (transducin) takes place at Meta II stage, 11-cis-retinal attached to rhodopsin is regarded as an inverse agonist while all-trans retinal attached to Meta II can be regarded as a full agonist. Since the same chromophore of rhodopsin changes from an inverse agonist to a full agonist upon illumination of light, its conformational changes can be studied by observing changes in absorption spectrum.
The conversion of rhodopsin to Batho is a rapid process that takes place within 200 fs. Each conformational change leading to Meta II takes about a few milliseconds, which is long enough to allow a protein to undergo a significant conformational change involving spatial displacement of the secondary structures of the protein. It has been shown that the conformational change of opsin causes the beta-ionon moiety of the retinal chromophore to change its direction from the 6th helix (TM6) to the 4th helix (TM4) (Bohan, B. et al., Science, 288, 2209-2212 (2000)). This implies that the arrangement of helices has been altered as a result of photoisomerization.
Also, Khorana and Hubbell in their experiment illuminated light onto a mutant rhodopsin, which has been spin-labeled in a site-directed manner by taking advantage of SH groups in the mutant site-specifically substituted with cysteine, and demonstrated that the conformational changes of rhodopsin to Meta II are accompanied by conformational changes of the intracellular loops and helices. They proposed a model in which the entire TM6 helix undergoes significant rotation. The model implies considerable conformational changes of membrane proteins (Farrens, D. L. et al., Science 274, 768-770 (1996)).
Light energy absorbed by the chromophore is harnessed to cause initial conformational change of opsin. Transition to the final active form, the Meta II conformation, begins with proton transfer from the protonated Schiff base to its counterion, Glu 134 in TM3, to form neutral Schiff base. The neutralization of the Schiff base allows movement of the helix and, ultimately, the rotation of TM6, causing the shift to the Meta II conformation.
Of the different photoactivated intermediates of rhodopsin, the final Meta II conformation has proven to be the only form that has been fully activated (Khorana, H. G. J. Biol. Chem., 267, 1-4 (1992)). However, opsin without the chromophore is known to exhibit approximately 5% activity, and mutant opsin in which Glu134, which serves as a counterion of the protonated Schiff base, has been substituted with Gln exhibits approximately 50% activity even in the absence of the chromophore.
This mutant opsin is known to be deactivated when 11-cis-retinal is added and irradiation with light converts it to all-trans-retinal, which in turn is converted to fully activated Meta II conformation. Thus, it has been shown that opsin has several active forms (Kim, J.-M. et al., Proc. Natl. Acad. Sci. USA, 94, 14273-14278 (1997)).
It is also known that G-protein (transducin) does not bind opsin when rhodopsin is in its Meta I state while it binds opsin without activating it when rhodopsin is in its Meta Ib state (Tachibanaki, S. et al., Biochemistry 36, 14173-14180 (1997)).
As described, a series of events, including conformational changes of opsin and its interaction with G-protein, and subsequent activation of G-protein, take place over the course of the process from Lumi to Meta II. During this process, the rotation of TM6, essential for the activation of rhodopsin, provides the G protein-coupled receptor with the structural specificity required for ligand recognition. Specifically, it has been shown that the amino acid residues in the ligand binding site involved with TM6 before the rotation of TM6 are different than the ones involved with TM6 after the rotation of TM6, and amino acid residues that serve to recognize full agonists are different than those that serve to recognize antagonists.
In fact, mutants are often found in which alteration of some of the amino acid residues in TM6 affects the binding of full agonists but not the binding of antagonists. Such phenomenon will be explained by taking into account the conformational changes of the receptors.
Studies on conformational changes of rhodopsin suggested that the arrangement of TMs is significantly different between the receptors that bind antagonists and the receptors that bind agonists. For this reason, the crystal structure of rhodopsin does not solely provide a structural model for every receptor/ligand complex.
A comparison between the crystal structure of rhodopsin and a structural model for Meta II in accordance with the present invention is shown in FIG. 2. The significant displacement of highly conserved Trp265 in TM6 suggests that different amino acid residues are involved in recognizing agonists and antagonists.
As described above, several experiments demonstrated that photoactivation of rhodopsin brings about conformational changes of opsin (See, for example, Farrens, D. L. et al., Science 274, 768-770 (1996). Kim, J.-M. et al., Proc. Natl. Acad. Sci. USA, 94, 14273-14278, (1997)). Nonetheless, the nature of specific conformational change has yet to be understood.
Accordingly, it is an objective of the present invention to simulate three-dimensional structures of these photoactivated intermediates of rhodopsin by means of computer graphics and scientific calculation and to thereby construct structural models for their complexes formed with ligands (chromophores) that can bind rhodopsin as well as three-dimensional structural models for the activated rhodopsin intermediates in the structural models of the complexes.
It is another objective of the present invention to provide a method for identifying, screening for, searching for, or evaluating whether a given ligand is a full agonist, a partial agonist, an antagonist, or an inverse agonist by constructing three-dimensional models for general G protein-coupled receptors (GPCRs) other than rhodopsin from the three-dimensional structural models for the activated intermediates of rhodopsin and, for each of the three-dimensional models, constructing structural models for their complexes formed with ligands and analyzing the interaction of GPCRs with corresponding ligands. It is still another objective of the present invention to provide a method for designing a novel ligand molecule that acts as an agonist or an antagonist of a GPCR.